1. Technical Field
The present invention relates to a method of identifying genes based on the gene's function. More specifically, the present invention relates to a device for performing identification of genes that are upstream regulators of the expression of other genes.
2. Description of the Related Art
The Human Genome project led to the elucidation of the sequence of human genes, but the function of many of the genes remains unknown. The current methods of discovering gene function require laborious work and can take months to accomplish. This current approach to the study of gene function is based on the identification of genes whose expression is altered in a disease state, or as a result of a given stimulus. There are cases in which the level of expression of a gene is unaltered, but the gene's function is crucial for a biochemical process associated with a stimulus or a disease. These genes can not be detected by the methods currently used by those of skill in the art. Additionally, the attempt to define drug targets according to changes in levels of expression of genes leads to false negatives because the substrates of many known drugs are proteins that are expressed at constitutive levels. Individuals in the art therefore began to develop alternative methods for determining the function of genes, as are described below.
Alternative methods were developed for inactivating the expression of genes based on knowledge of the gene's sequence. The process of inactivating genes by these methods is referred to as gene silencing. The gene silencing methods can be used to facilitate the functional screening of genes. Two of the methods of silencing genes are the antisense method and RNA interference.
Inactivation of most genes does not lead to visible phenotypic changes in cell cultures expressing those genes. Knowledge about the function of genes whose silencing does not lead to obvious changes in phenotype can be derived from information concerning other genes that have been activated or repressed as a result of silencing a given gene. Important information relating to gene function includes, but is not limited to: (1) data regarding which other genes are regulated by a gene of interest, namely; which genes are activated, induced, or repressed as a result of silencing the gene of interest (i.e., which genes are regulated downstream of the gene of interest); and (2) data regarding which other genes regulate the expression of a given gene of interest (i.e., which genes are upstream regulators of the gene of interest).
Sequitur, Inc, of Natick, Mass., offers a product called “omniscreen.” The “omniscreen” method involves inactivating specific genes on high-throughput cell culture disease model screens (phenotypic screens) using a library of antisense compounds targeting thousands of target genes selected on the basis of homology to drugable target gene families. During the process, the Sequitur, Inc., library of antisense targets is used. An alternative method to gene silencing by antisense oligonucleotides, is silencing by RNA interference. For example, in Caenorhabditis elegans, the injection of double-stranded RNA (dsRNA) resulted in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). The same result was found by feeding worms with bacteria that express dsRNA gene (Maeda I. et al.). The term RNAi was coined after the discovery that the injection of dsRNA into C. elegans interferes with the expression of specific genes highly homologous in sequence to the delivered dsRNA (Fire A. et al).
Similar approaches to the “omniscreen” method are disclosed in the publications by Schmid et al. Followed by the studies in plants and invertebrates that have showed that the use of dsRNA of approximately 21–25 nucleotides function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing RNA interference recently the same effect was observed in mammalian cells (Elbashir, S. M. et al. and Caplen N. J. et al, and by Harborth et al.). Both Schmid and Harborth (suggested using RNAi to screen for genes that are essential for cell viability and maintenance of a visible phenotype. The genes were classified based on the impaired cell growth following RNAi.
When combined with microarrays (Pomeroy et al. Mimics K et al, Mungall A J. and Mills J C, et al and Ziauddin J) the above described methods are efficient methods that can be used to identify genes that are regulated by a given gene (i.e., genes that are downstream regulators of the gene of interest). However, there are no high throughput methods that can be used to identify upstream regulators of a given gene. It would therefore be useful to develop a method for high-throughput identification of upstream regulators of a gene. There are also genes whose expression can serve as early markers for diseases and other pathological and physiological processes. It is of particular interest to rapidly identify many genes that are upstream regulators of these genes, and the relationships among these upstream regulators.